The present invention relates to “patch clamping” for investigating ionic and molecular transport through cellular membranes via ion channels and, in particular, to a substrate providing a set of nano to microscale pores that may be readily sealed to cellular membranes.
The lipid bilayers that make up cell membranes include ion channels that control the flow of ions into and out of cells. Certain ion channels open in response to signaling molecules including naturally occurring signaling molecules and drug molecules. In the development of therapeutic drugs, it is necessary to determine the effect of the drug on ion channels either to avoid adverse effects or to create a positive therapeutic effect for the treatment of ion-channel related diseases.
Analysis of the response of ion channels may be conducted with a so-called “patch clamp”, traditionally a micropipette adhered to the surface of a cell by a slight suction. An electrical connection to the interior of the cell can be made, for example, by applying a sharp suction pulse to the pipette to open a hole in the cell wall. Measurement of small electrical changes across the cell membrane may then be used to deduce the opening or closing of particular ion channels.
The small amount of electrical current involved in these measurements requires an extremely high resistance seal between the pipette and the cell wall (a gigaohm seal or gigaseal). Typically a gigaohm seal should be of the order of 15-20 gigaohms and at least 5 gigaohms.
Drug screening can require a large number of ion channel measurements. Accordingly, in current practice, the pipette can be replaced with a plate having multiple small pores each of which may accept a cell. The plate array allows the parallel processing of multiple cells and may be more readily integrated into automated equipment than a pipette.
The production of nanoscale holes in a plate structure is relatively difficult. One technique requires irradiating a glass or quartz substrate with heavy ions which leave behind a track of molecular damage that may preferentially be etched, for example, with hydrofluoric acid. The timing of the etch is controlled so that it breaks through on the far side of the substrate to produce the correct hole size.
The need for access to heavy ion accelerators for the production of nano-sized holes can be avoided by a second technique which employs a laser to ablate a crater through a pre-thinned glass substrate. By controlling the duration and power of the laser, a generally conical crater may break through the opposite side of the substrate with an appropriate size of hole.
One disadvantage to the laser approach is that it spreads molten glass and debris on the exit of the hole. This debris impedes proper adherence between the cell membrane and the lip of the hole leading to a reduction in the electrical resistance of the hole so formed.